Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged.
Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, "Careina Edgooms" <[email protected]> wrote: > Dear CCP4 board > > I have a protein that exists in equilibrium between monomer and dimer and > I'm trying to calculate KD using size exclusion. The problem is that the > column dilutes my sample so that if I put 20 uM on to the column, I only > recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting > confused as to how to plot my KDs. Do I not regard my initial > concentrations at all and work only with the final concentrations that come > off the column? > I would plot [monomer] squared vs [dimer] and I will assume that the ratio > of monomer to dimer will stay constant as the protein passes through the > column. (also I would calculate [dimer] using 2x monomer extinction > coefficient) > > Does this seem a reasonable way to calculate KDs and reasonable argument? > Also I am looking for good references for calculating Kds when dealing with > dimerization > Thanks and sorry for off topic question > Careina >
